scholarly journals Single-Cell Transcriptional Landscapes of Human Bone Marrow Reveal Distinct Erythroid Phenotypes Underpinned By Genotype in Diamond-Blackfan Anemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 1-2
Author(s):  
Deena Iskander ◽  
Guanlin Wang ◽  
Elisabeth F Heuston ◽  
Chrysi Christodoulidou ◽  
Bethan Psaila ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Single Cells ◽  
Developmental Trajectory ◽  
Gene Set Enrichment Analysis ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia

Background: Diamond- Blackfan Anemia (DBA) is a rare, heritable ribosomopathy caused by mutations in ribosomal protein large (RPL) and small (RPS) subunit genes. The diagnostic criteria of DBA include presentation in infancy with virtually no mature erythroblasts (EB) on bone marrow (BM) examination, however atypical presentations in later life with milder haematological manifestations are increasingly reported. The cellular and molecular mechanisms underpinning variable clinical phenotypes, and how they relate to genotype, are yet to be elucidated. Furthermore, as many DBA patients do not respond to corticosteroids, understanding the pathological processes contributing to erythroid failure is a prerequisite for developing novel, precision-medicine therapies. Aim: To elucidate phenotypic and functional differences in erythropoiesis in RPS- and RPL-DBA, using primary BM samples from patients. Methods: We performed single-cell transcriptome profiling (scRNAseq), using the 10X Genomics chromium platform, of 45,488 CD34+ Lineage negative (Lin-) BM hematopoietic stem and progenitor cells (HSPC), purified by fluorescence-activated cell sorting. We included six patients with red cell transfusion-dependent DBA (aged 2-19y) with mutations in RPS19 (n=3), RPL11 (n=1) and RPL5 (n=2) and three healthy donors (aged 3-17y). We validated our findings using bulk RNAseq, functional assays, and deep immunophenotyping-based dissection of the haematopoietic architecture of DBA BM ex vivo. Results: High quality sequencing data was obtained for all nine donors; after quality control, 41,415 single cells were carried forward for analysis. Unsupervised clustering analysis and lineage identification revealed two divergent cellular patterns in DBA compared with age-matched control BM that segregated with genotype: a selective loss of erythromegakaryocyte (E/MK) progenitors in RPS-DBA, but relative preservation of the erythroid developmental trajectory in RPL-DBA, at the expense of megakaryopoiesis (Fig 1a). Gene Set Enrichment Analysis (GSEA) of differentially expressed genes between control and DBA HSPC clusters revealed p53-mediated apoptosis, TNFa-, IFNa- and IFNg-mediated inflammatory pathways in DBA EP. Although these pathways were enriched across all HSPC populations irrespective of genotype, they were detected at an earlier stage of the stem cell hierarchy, and more potently, in RPS- versus RPL-EP. Expression of transcriptional targets of the master E/MK transcription factor, GATA1, was significantly upregulated in RPL- versus RPS-DBA EP (Fig 1b), supporting the maintained erythroid program in RPL-DBA HSPC. However, expression of genes denoting erythroid differentiation, including adult haemoglobin (Hb) genes, was significantly elevated in RPL-DBA, suggesting aberrant accelerated differentiation to EB. These findings were confirmed by immunophenotypic examination of patient BM and bulk RNAseq of RPL-EB. Additionally, single-cell clonogenic assays of RPL-DBA EP showed severe qualitative defects. Thus, although erythroid commitment occurs in RPL-DBA, it is coupled with accelerated maturation beyond EP to functionally impaired mature EB, enriched in inflammatory and p53 pathways. Finally, we analysed the clinical characteristics of the 161 patients comprising the U.K DBA registry. In line with the milder erythroid specification defect, patients with RPL-DBA (n=44) presented with anaemia later (P=0.004), and with a higher average Hb concentration (P=0.04), than those with RPS-DBA (n=63). Furthermore, we identified higher corticosteroid responses in RPL-DBA, assessed at 6 months post initiation (P=0.006), consistent with our findings in RPL-DBA of preservation of the cellular EP populations that are targeted by corticosteroids. Impact: In conclusion, a preserved but distinct erythroid developmental trajectory, characterised by accelerated differentiation, underpins a milder haematological phenotype in RPL-DBA. Furthermore, we reveal the first single-cell transcriptomic dataset from haematopoietic cells in a ribosomopathy, uncovering novel cell intrinsic and extrinsic pathogenetic insights into failing erythropoiesis in DBA. Integration of these data with clinical genomics and phenomics provides a paradigm by which single cell approaches can be used to decipher genotype-phenotype relationships in Mendelian genetic disorders. Disclosures Mead: Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; Celgene/BMS: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding; Abbvie: Consultancy; CTI: Consultancy; Gilead: Consultancy.

scholarly journals Single Cell RNA-Seq Reveals Deranged Developmental Hierarchy with Coexisting Oncogenic States and Immune Evasion Programs in ETP T-ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3953-3953
Author(s):  
Amy Guillaumet-Adkins ◽  
Praveen Anand ◽  
Huiyoung Yun ◽  
Yotam Drier ◽  
Anna Rogers ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Single Cells ◽  
Pearson Correlation ◽  
Normal Donor ◽  
Developmental Hierarchy

Introduction: Early T-cell precursor acute lymphoblastic leukemia (ETP T-ALL) is a distinct subtype of T-ALL characterized by higher rates of relapse and induction failure. Large-scale genetic sequencing studies have identified frequently mutated oncogenes and gene fusions in ETP T-ALL, while bulk transcriptome analyses have revealed expression features resembling myeloid precursors and myeloid malignancies. However, the contributions of intra-tumoral functional heterogeneity and microenvironment to tumor biology and treatment failure remain unknown. Methods: We performed full-length single-cell RNA-sequencing of 5,077 malignant and normal immune cells from bone marrow or blood from five patients with relapsed/refractory ETP T-ALL (based on immunophenotyping, all with NOTCH1 mutations), before and after targeted therapy against NOTCH1. These patients were enrolled on a phase I trial with the γ-secretase inhibitor (GSI) BMS-906024 (NCT01363817). Expression of selected genes was validated by RT-PCR, flow cytometry and immunohistochemistry. Results: Single cell transcriptome analyses revealed a deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cells implying ineffectual commitment to either lymphoid or myeloid lineage. Most ETP T-ALL cells co-expressed HSC (hematopoietic stem cell), CMP (common myeloid progenitor) and CLP (common lymphoid progenitor) signatures simultaneously (Pearson correlation: CLP-CMP: R= 0.41, p < 2.2e-16; HSC-CLP: R= 0.53; p < 2.2e-16; HSC-CMP: R = 0.39, p <2.2e-16). Only a fraction of cells (less than 15%) demonstrated mutually exclusive CLP or HSC signatures. In contrast, CLP, CMP and HSC signatures were not co-expressed and always negatively correlated in normal bone marrow cells (CLP-CMP: R= -0.11, p < 2.2e-16; HSC-CLP: R= -0.38; p < 2.2e-16; HSC-CMP: R = -0.67, p <2.2e-16). Direct targeting of NOTCH1 as the driving oncogene has shown disappointing results in the clinical setting due to the rapid development of resistance. PI3K activation has been shown as a genetic mechanism of Notch resistance, however it is unclear if transcriptional rewiring can give rise to PI3K dependent cells after Notch inhibition. To address this question, we predicted the activity of signaling pathways in single cells after Notch inhibitor treatment using PROGENy. Most single cells demonstrated loss of Notch signaling. PI3K signaling activity was the most anti-correlated signaling pathway to Notch signaling (Pearson correlation: R= -0.51, p < 2.2e-16). Of note, this population preexisted at a frequency of ~30% in the untreated population, coexisting with cells with high Notch activation. Analysis of the immune microenvironment revealed an oligoclonal T-cell population in ETP T-ALL compared to normal donor T-cells. CD8+ T-cells from ETP patients expressed markers of T-cell exhaustion (PDCD1, TIGIT, LAG3, HAVCR2). Analyses of expression levels of the respective ligands on leukemic blasts and the predicted interaction with their receptors on endogenous CD8+ T-cells demonstrated the highest interaction score between HAVCR2 and its ligand LGALS9. LGALS9 was universally expressed in all leukemic cells, which was confirmed by flow cytometry staining in leukemic blasts and IHC staining in bone marrow of 8 patients with ETP T-ALL and 7 patients with T-ALL. T-ALL supernatant increased expression levels of the exhaustion markers HAVCR2,TIGIT and decreased effector marker GZMB in polyclonal activated normal donor CD8+ T-cells (RT-PCR). This effect was abrogated by neutralizing LGALS9 and could be rescued with recombinant LGALS9. Conclusion: We identified deranged developmental hierarchy characterized by co-expression of stemness programs in multiple malignant cell states and ineffectual commitment to either lymphoid or myeloid lineage in ETP T-ALL. Leukemic blasts demonstrate preexisting heterogeneity of diverse oncogenic states as evidenced by opposing PI3K and Notch activity, suggesting possible novel combination therapies. Notch inhibition abolishes the Notch high state without effecting the PI3K active state. Finally, we demonstrate a possible role for HAVCR2-LGALS9 interactions in causing CD8+ T-cell dysfunction in ETP T-ALL patients, which may provide a novel therapeutic strategy in this disease. Disclosures Silverman: Takeda: Consultancy; Servier: Consultancy, Research Funding. Lane:AbbVie: Research Funding; Stemline Therapeutics: Research Funding; N-of-One: Consultancy. DeAngelo:Glycomimetics: Research Funding; Amgen, Autolus, Celgene, Forty-seven, Incyte, Jazzs, Pfizer, Shire, Takeda: Consultancy; Blueprint: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Abbvie: Research Funding. Lohr:Celgene: Research Funding; T2 Biosystems: Honoraria.

scholarly journals Single-Cell RNA-Seq Reveals the Differentiation Hierarchy of Normal Human Bone Marrow and a Distinct Transcriptome Signature of Monosomy 7 Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 574-574
Author(s):  
Xin Zhao ◽  
Shouguo Gao ◽  
Xingmin Feng ◽  
Delong Liu ◽  
Sachiko Kajigaya ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Single Cell ◽  
Signaling Pathway ◽  
Copy Number ◽  
Research Funding ◽  
Single Cells ◽  
Myeloid Cells ◽  
Healthy Controls ◽  
Monosomy 7

Abstract Monosomy 7 is a frequent cytogenetic abnormality in hematopoietic malignancies and a general indicator of poor prognosis. Due to lack of distinct cell surface markers between monosomy 7 cells and normal cells, it is not feasible to physically separate aneuploid from diploid cells. We performed single-cell RNA-seq (scRNA-seq), which allows the entire transcriptome of large numbers of single cells to be assayed in an unbiased way, to investigate hematopoietic differentiation of normal and aneuploid human hematopoietic cells. Bone marrow samples were collected from four patients (P1-P4) with myelodysplastic syndrome, and four healthy volunteers. Conventional cytogenetics showed -7/7q- in bone marrow cells from P1, P3 and P4, and dup(1)(q21q32) in cells from P2; retrospectively, P2 was found positive for monosomy 7 as well as trisomy 8 by fluorescence in situ hybridization. Fresh CD34+CD38- and CD34+CD38+cells were sorted by flow cytometry and then subjected to Fluidigm C1 Single-Cell Auto Prep System for scRNA-seq. After excluding cells with low transcriptome coverage, 326 cells from P1 and P2 (analysis is in progress for P3 and P4), and 391 cells from healthy subjects were analyzed by comparison of transcriptomes from 17,071 genes. Nonlinear dimension reduction and visualization were achieved using t-distributed Stochastic Neighbor Embedding (tSNE). Cells from healthy controls clustered into seven subgroups based on their gene expression pattern, and each group could be associated with a previously reported hematopoietic cell type by known marker genes (Laurenti E, Nat Immunol, 2013). These cell types included hematopoietic stem cell (HSC), multilymphoid progenitor (MLP), granulocyte-monocyte progenitor (GMP), Pro-B cell (ProB), earliest thymic progenitor (ETP), and megakaryocytic-erythroid progenitor (MEP) (Fig 1a). Individual cells from healthy controls were ordered by Monocle software based on their expression profile similarity to uncover a differentiation hierarchy. A two-branch trajectory of development from HSC was revealed, with one branch progressing towards erythroid cell and the other to lymphoid/myeloid cells (Fig 1b). This pattern differs from the classic hematopoietic model, but is consistent with reports claiming existence of early-lymphoid-biased progenitors that retain myeloid but not erythroid potential (Doulatov S, Nat Immunol, 2010), and of dominance of multipotent and unipotent progenitors over scarce oligopotent progenitors in the adult marrow hematopoietic hierarchy (Notta F, Science, 2016). We compared single cells from patients and healthy controls for regional and chromosomal copy number differences in gene expression. We identified subclonal populations of cells from patients that showed decreased expression of chromosome 7 genes (60% in P1, and 55% in P2; Fig 1c and 1d), and increased expression of chromosome 8 (77% in P2) and chromosome 1 long arm genes (P2), at FDR=0.05 estimated with cells from health donors. Gene Ontology enrichment analysis using topGO indicated that cells with low global expression of chromosome 7 genes had dysregulated expression of immune related genes, including B cell receptor signaling pathway, T cell activation and differentiation, antigen receptor-mediated signaling pathway, as well as signal transduction and Fc-γ Receptor signaling pathway. ScRNA-seq analysis reveals a simple pattern of normal human hematopoietic development and the molecular signature of aneuploid cells from patients with developing "clonal evolution". This powerful method should improve characterization of functional changes in human cells with chromosome abnormalities. Figure 1 a. Single-cell gene expression patterns assigned single cells from healthy controls to seven clusters. 38N: CD34+CD38- population; 38P: CD34+CD38+ population. Different shapes represent cells from different subjects. b. Pseudo-time ordering of cells using Monocle reveals a two-branch stepwise development from stem cells to erythroid or lymphoid/myeloid cells. c. Heatmap of the copy-number variation (CNV) signal normalized against healthy controls shows CNV changes by chromosome (columns) for patients' individual cells (rows). d. Genome-wide gene expression binned per chromosome in single cells from P1, P2 and healthy controls. Chromosomal mapping reads values were median centered. Figure 1. a. Single-cell gene expression patterns assigned single cells from healthy controls to seven clusters. 38N: CD34+CD38- population; 38P: CD34+CD38+ population. Different shapes represent cells from different subjects. b. Pseudo-time ordering of cells using Monocle reveals a two-branch stepwise development from stem cells to erythroid or lymphoid/myeloid cells. c. Heatmap of the copy-number variation (CNV) signal normalized against healthy controls shows CNV changes by chromosome (columns) for patients' individual cells (rows). d. Genome-wide gene expression binned per chromosome in single cells from P1, P2 and healthy controls. Chromosomal mapping reads values were median centered. Disclosures Desierto: GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding. Young:Novartis: Research Funding.

scholarly journals Loss of Abelson Interactor-1 Is Linked to Inflammatory Hematopoiesis and Accelerated Aging of Hematopoietic System

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1285-1285
Author(s):  
Anna Dorota Chorzalska ◽  
John Morgan ◽  
Max Petersen ◽  
Diana O Treaba ◽  
Adam J Olszewski ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Animal Models ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Bone Marrow Failure ◽  
Accelerated Aging ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Oncology Research

Abstract Background: Hematopoietic stem cells (HSC) ensure homeostasis and lifelong maintenance of hematopoietic system, but with age, they gradually lose quiescence, self-renewal potential, and system restoration capacity. HSC aging results in a differentiation shift towards myeloid lineage, anemia, thrombocytosis, decrease in T and B cells, imbalance in macrophage function, and increased osteoclast activity. Mechanisms involved in HSC aging include increased mTOR activity and ROS production, impaired autophagy, epigenetic reprograming, and cumulative DNA damage. Intriguingly, cellular and molecular similarities between aging and inflammation have led to a novel concept of "inflammation-associated aging of hematopoiesis". Understanding the molecular mechanisms responsible for this process may impact strategies targeting age-related diseases, including neoplasms. However, to date only few primary animal models of inflammation have shown bone marrow failure, so new animal models need to be established to provide mechanistic insight into the long-term implications of chronic inflammation on the hematopoietic system. We have previously shown that bone marrow-specific deletion of an adapter protein Abelson interactor-1 (Abi1) leads to a myeloproliferative neoplasm (MPN)-like disease in 35-56-week-old mice, mechanistically associated with increased activity of Src Family Kinases (SFKs), STAT3 and NF-κB. At both transcript and protein levels, Abi-1 is also significantly reduced in HSCs and granulocytes from patients with primary myelofibrosis (PMF), and Abi-1-deficient HSC in human PMF show increased SFK-STAT3-NF-κB signaling (Chorzalska, ASH 2017). Methods: Myeloid/lymphoid, stem/progenitor populations profiling by FACS, bone marrow transplantation assays, transcriptomics and proteomics analyses as well pro-inflammatory cytokine profiling and histopathology analyses were performed on the transgenic Abi-1KO mice carrying bone marrow-selective knockout at 4 weeks post-recombination, upon confirming both inducible inactivation of the Abi1flox allele and loss of Abi-1 protein in the marrow (Fig.1A, B). Results: To better understand initial systemic events that lead to the development of MPN-like disease in aged Abi-1KO mice we have now characterized early changes within the hematopoietic system associated with loss of Abi-1. Blood count analysis indicated leukocytosis, anemia and thrombocytosis, and an increase in the fraction of myeloid (CD11b+/Gr-1+) as well as macrophage/monocyte (F4/80+) cells at the expense of lymphoid (B220+) cells in Abi-1KO relative to Abi-1WT mice (Fig.1C). Previously reported 2.6-fold increase in Abi-1KO LT-HSCs (Chorzalska, ASH 2017) was now shown to be associated with 30% increase in number of LT-HSCs is in the S/G2/M phases of the cell cycle relative to Abi-1WT LT-HSCs (Fig. 1D). Lethally irradiated recipient C57BL/6 wild-type mice transplanted with bone marrow cells from Abi-1KO relative to Abi-1WT mice (in the absence of competitor cells) showed progressive loss of chimerism in primary and secondary recipients (Fig. E). Genome-wide gene expression analysis of Abi-1WT vs. Abi-1KO LSK cells showed significant overexpression of genes regulated by or involved in regulation of the NF-κB pathway (Fig. 1F). Plasma cytokine levels showed 2-fold increase in IL-1B, IL-12, IL-17, IL-23, IL-27, and MCP-1 and nearly 10-fold increase in INFγ (Fig. 1G). Label-free, intensity-based quantitative proteomic analysis of bone marrow from 20-week-old Abi-1KO and Abi-1WT mice showed abundance of peptides derived from Mac-1, myeloperoxidase, STAT1, STAT3, and SFKs - Hck and Fgr, confirming not only activation of SFKs and STAT3 signaling, but also increase in proteins associated with myeloid lineages (Fig. 1H). Loss of bone density (Fig. 1I) and a significant decrease in thymus size (Fig. 1J) were observed in Abi-1KO mice relative to Abi-1WT mice. Conclusions: In sum, phenotypic analyses performed 4-10-weeks post Abi1 gene inactivation indicate changes consistent with accelerated aging of hematopoietic system that are mechanistically linked to inflammatory SFK-STAT3-NF-κB signaling. To our knowledge this is the first animal model linking accelerated inflammation-driven aging of hematopoietic system to development of an MPN in aged mice. Disclosures Olszewski: Genentech: Research Funding; TG Therapeutics: Research Funding; Spectrum Pharmaceuticals: Consultancy, Research Funding. Reagan:Pfizer: Research Funding; Alexion: Honoraria; Takeda Oncology: Research Funding.

scholarly journals Single-Cell Multi-Omics in Human Clonal Hematopoiesis Reveals That DNMT3A R882 Mutations Perturb Early Progenitor States through Selective Hypomethylation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 1-2
Author(s):  
Anna S Nam ◽  
Neville Dusaj ◽  
Franco Izzo ◽  
Rekha Murali ◽  
Tarek H Mouhieddine ◽  
...  
Keyword(s):  
Single Cell ◽  
Research Funding ◽  
Gene Set Enrichment Analysis ◽  
Driver Mutations ◽  
Hematopoietic Stem ◽  
Binding Motifs ◽  
Mutation Status ◽  
Clonal Hematopoiesis ◽  
Cd34 Cells ◽  
Myeloid Progenitors

Leukemia driver mutations have been identified in clonal hematopoiesis (CH; Jaiswal et al, NEJM, 2014). This provides a window of opportunity to interrogate the downstream impact of driver mutations in the earliest stages of neoplasia, before the accumulation of additional drivers that lead to frank malignancy. However, CH mutated cells are morphologically and phenotypically similar to normal cells. Thus, previous characterization in primary human samples have largely focused on genetic identification of these clonal outgrowths. To overcome this limitation and define the downstream effects of CH mutations, we leveraged multi-omic single-cell sequencing to profile the mutation status and whole transcriptome for the same cells at high-throughput (GoT; Nam et al, Nature, 2019). To identify CH samples, we screened 136 stem cell grafts collected for autologous transplant from multiple myeloma patients in remission (Mouhieddine et al, Nat Comm, 2020). We identified four individuals with DNMT3AR882 mutations with VAF &gt; 0.05, allowing the profiling of 27,324 CD34+ cells (Fig. 1a-b). DNMT3A R882 mutations in these human CH samples did not result in a significant differentiation block in the hematopoietic stem cells, as assessed by pseudotime analysis (Fig. 1c). However, specific differentiation skews were apparent in multi-lineage progenitors, corresponding to the highest expression of DNMT3A in mutated immature myeloid progenitors (IMP, i.e. common myeloid progenitors/granulocyte-monocyte progenitors, Fig. 1d). We observed the expansion of mutated IMPs (Fig. 1e) that showed priming toward the megakaryocytic-erythroid (ME) lineage (Fig. 1f). DNMT3AR882 lympho-myeloid primed progenitors showed a myeloid bias and disfavored lymphoid development (Fig. 1g). In these CD34+ progenitors, we identified dysregulated expressions of megakaryocytic lineage markers, such as CD9 and PLEK, consistent with the ME-bias (Fig. 1h). We also observed downregulation of lymphoid differentiation gene ZBTB1, corresponding to the myeloid over lymphoid skewing (Fig. 1h). Finally, we identified dysregulated expression of genes involved in TNF-alpha signaling (e.g. TNFRSF4, TNFSF13B), suggesting a pro-inflammatory state. To identify activated pathways, we performed a gene set enrichment analysis that revealed activation of MYC and IL-6 signaling (FDR &lt;0.05, Hallmark). A focused analysis of regulatory networks identified enhanced activity of key transcription factors (TFs) involved in ME differentiation such as GATA1 and FLI1, as well as those involved in inflammation such as NFKB/REL (FDR &lt;0.05). We also identified increased expression of polycomb repression complex 2 (PRC2) target genes (FDR &lt;0.05). To determine how aberrant DNA methylation may serve as a link between DNMT3A mutations and the observed transcriptional dysregulation, we performed single-cell multi-omics that simultaneously profiles the cells' methylome and transcriptome, linked with mutation status (Gaiti et al, Nature, 2019; Fig. 1i). By comparing the methylation rates in mutated vs. wildtype CD34+ cells within the same sample, we found that DNMT3AR882 result in selective hypomethylation that preferentially impacts CpG islands (Fig. 1j) and PRC2 targets (Fig. 1k), which may underlie the dysregulated expression of PRC2 targets in DNMT3AR882 progenitors. Notably, DNMT3AR882-induced hypomethylation was enriched in a specific sequence context in which the CpG is followed by a T nucleotide (Fig. 1l). This DNMT3AR882 motif was enriched in the DNA binding motifs of central hematopoietic TFs, including MYC/MAX (Fig. 1m), suggesting that DNMT3AR882-induced hypomethylation may serve as a mechanism to preferentially increase the activity of these TFs. Consistent with this hypothesis, joint single-cell methylome and transcriptome data revealed that the expression of MYC targets increased with hypomethylation of its binding motifs (Fig. 1n), providing a mechanism of enhanced MYC activity due to DNMT3AR882 mutations. Altogether, we report the direct examination of the consequences of DNMT3AR882 mutations in primary CD34+ cells in CH. We discovered that DNMT3A is most highly expressed in mutated multi-lineage progenitors, promoting their expansion and biasing their downstream differentiation. This is accompanied by a disruption to differentiation through PRC2 and MYC target reactivation via selective hypomethylation. Disclosures Abdel-Wahab: Envisagenics Inc.: Current equity holder in private company; H3 Biomedicine Inc.: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy. Ghobrial:Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Karyopharm Therapeutics: Consultancy, Honoraria; Cellectar: Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Novartis: Consultancy; Noxxon Pharma: Consultancy; Genentech: Consultancy; GlaxoSmithKline: Consultancy; GNS Healthcare: Consultancy; AbbVie: Consultancy. Landau:Bristol Myers Squibb: Research Funding; Illumina: Research Funding.

Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single Cell RNA Sequencing Integrated with Highly Sensitive Genotyping Method

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-34
Author(s):  
Masahiro Marshall Nakagawa ◽  
Ryosaku Inagaki ◽  
Yutaka Kuroda ◽  
Yasuhito Nannya ◽  
Lanying Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Simultaneous Detection ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Single Cell Sequencing ◽  
Clonal Hematopoiesis

Background Recent evidence suggests that age-related clonal hematopoiesis (CH) might represent the earliest precursor of myeloid neoplasms. Although the exact mechanism of clonal selection that shapes CH is still to be elucidated, both cell intrinsic and non-cell intrinsic effects of mutations, including the interplay between mutated cells and the bone marrow environment, are thought to play important roles, which are best studied using single-cell sequencing analysis of both mutations and gene expression. Methods We performed single-cell sequencing of hematopoietic stem and progenitors (HSPCs) from BM of the 16 patients with CH along with 16 control patients without CH identified by screening otherwise healthy individuals who received hip joint replacement, using a novel platform that enables simultaneous detection of gene mutations and expression based on the Fluidigm C1-HT system. Sequence data were analyzed with Seurat (Stuart et al Cell 2019) with integration of genotyping information. Cells were clustered and each cluster was assigned by marker-gene expressions for major cell-types in HSPCs, including hematopoietic stem cell (HSC)-like and erythroid progenitors. Cells were grouped by their genotypes and pathway analysis were performed. Results In total, we identified 35 subjects who had CH-related mutations, including those affecting DNMT3A, TET2, ASXL1, SF3B1, PPM1D, IDH1, GNB1 and TP53, of which 11 had more than one CH-related mutation. Most of these mutations showed a low variant allele frequency (VAF) ≤ 0.05. However, clones having double mutations of DNMT3A/TET2 or those having biallelic TET2 mutations tended to show a higher VAF as high as 0.4, suggesting an enhanced clonal advantage for clones having multiple mutations. Using our novel single-cell platform, we analyzed 3,767 cells from control patients without CH and 1,474 mutated cells and 7,234 wild-type (WT) cells from patients with CH. By targeting both genomic DNA and RNA, we successfully obtained a sufficient number of single-cell reads for genes whose expression was too low to evaluate by only targeting RNA, such as TET2 and DNMT3A. Although some clones having a high-VAF mutation caused a skewed clustering to be detected as a CH clone, many clones with low-VAF mutations did not make distinct clusters, indicating the importance of genotyping at a single cell level to identify and characterize mutated cells. Simultaneous detection of genotype and expression allowed us to see the effect of CH-mutations on cell phenotype and differentiation. For example, cells having compound TET2/DNMT3A mutations were significantly enriched in the erythroid cluster, while another clone with double TET2 mutations were more enriched in the HSC-like cluster, compared to cells from individuals without CH (WTcont). These are in line with the previous findings of TET2/DNMT3A double knockout mice or TET2 knockout mice, respectively. In another case with an IDH1 mutation, IDH1-mutated (MUTIDH1) cells less contributed to the HSC-like fraction, showing an enhancement of cell proliferation-signature, compared to WT (WTIDH1) cells in the same patient. Strikingly, compared to WTcont cells, WTIDH1 cells were significantly enriched in the HSC-like fraction and showed an enhanced expression of cytokine-related pathway genes, which was in line with a finding seen in mouse cells treated with 2-hydroxy-glutalate, an mutant IDH-related oncometabolite. Similarly, when compared to WTcont cells, WT cells from patients with DNMT3A- (WTDNMT3A) or TET2- (WTTET2) mutated CH significantly showed an enhanced cell proliferation. HSC-like WTTET2 cells also showed aberrant IFN-response signatures compared to corresponding WTcont cells, which was confirmed in competitive transplantation of Tet2 heterozygous knockout (hKO) and WT cells in a mouse model; HSPCs of WT competitors transplanted with Tet2-hKO cells showed a significant enhancement of IFN-response signatures compared to those transplanted with WT cells. Intriguingly, monocytes of Tet2-hKO donors showed aberrant expression of S100a8/a9, which might contribute to the non-cell intrinsic effect of Tet2-hKO cells. Conclusions In CH, not only mutated cells but also surrounding WT cells show an aberrant gene expression phenotype, suggesting the presence of non-cell autonomous phenotype or an altered bone marrow environment that favors the positive selection of CH-clones. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Ogawa:Eisai Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Diamond-Blackfan Anemia: Erythropoiesis Lost in Ribosome Biosynthesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-2-SCI-2
Author(s):  
Stefan Karlsson ◽  
Johan Flygare ◽  
Pekka Jaako ◽  
David Bryder
Keyword(s):  
Bone Marrow ◽  
Platelet Count ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Macrocytic Anemia ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia

Abstract Abstract SCI-2 Diamond-Blackfan anemia (DBA) is a rare congenital erythroid hypoplasia that presents early in infancy. The classic hematologic profile of DBA consists of macrocytic anemia with selective absence of erythroid precursors in a normocellular bone marrow, normal or slightly decreased neutrophil, and variable platelet count. During the course of the disease some patients show decreased bone marrow cellularity that often correlates with neutropenia and thrombocytopenia. DBA is a developmental disease since almost 50% of the patients show a broad spectrum of physical abnormalities. All known DBA disease genes encode for ribosomal proteins that collectively explain the genetic basis for approximately 55% of DBA cases. Twenty-five percent of the patients have mutations in a gene encoding for ribosomal protein S19 (RPS19). All patients are heterozygous with respect to RPS19 mutations suggesting a functional haploinsufficiency of RPS19 as basis for disease pathology. Despite the recent advances in DBA genetics, the pathophysiology of the disease remains elusive. Cellular studies on patients together with successful marrow transplantation have demonstrated the intrinsic nature of the hematopoietic defect. DBA patients have a variable deficit in burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors. The frequency of immature hematopoietic progenitors in DBA patients is normal but their proliferation is impaired in vitro. Generation of animal models for RPS19-deficient DBA is pivotal to understand the disease mechanisms and to evaluate novel therapies. Several DBA models have been generated in mice or zebrafish. Although these models have provided important insights on DBA, they are limited in a sense that the hematopoietic phenotype and molecular mechanisms are likely to be influenced by the level of RPS19 downregulation. We have generated mouse models for RPS19-deficient DBA by taking advantage of transgenic RNAi. These models are engineered to contain a doxycycline-regulatable RPS19-targeting shRNA, allowing a reversible and dose-dependent downregulation of RPS19 expression. We demonstrate that the RPS19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count and the severity of the phenotype depends on the level of RPS19 downregulation. We show further that a chronic RPS19 deficiency leads to irreversible exhaustion of hematopoietic stem cells and subsequent bone marrow failure. Overexpression of RPS19 following gene transfer rescues the proliferative and apoptotic phenotype of RPS19-deficient hematopoietic progenitors in vitro, demonstrating that the phenotype is specifically caused by the RPS19 deficiency. Expression analysis of RPS19-deficient hematopoietic progenitors reveals an activation of the p53 pathway. By intercrossing the DBA mice with p53 null mice we demonstrate that inactivation of p53 in vivo results in a variable rescue of the hematopoietic phenotype depending on the level of RPS19 downregulation. Therefore, we conclude that increased activity of p53 plays a major role in causing the DBA phenotype but that other hitherto unidentified pathways also play a role, specifically in patients that have low levels of functional RPS19. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Neurotransmitter Receptor Gabbr1 Regulates Proliferation and Function of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3707-3707
Author(s):  
Adedamola Elujoba-Bridenstine ◽  
Lijian Shao ◽  
Katherine Zink ◽  
Laura Sanchez ◽  
Brian Cox ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Progenitor Cells ◽  
Gene Set Enrichment Analysis ◽  
Mouse Bone Marrow ◽  
Type I ◽  
Rna Seq ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
And Behavior

Hematopoietic stem and progenitor cells (HSPCs) have multi-lineage potential and can be used in transplants as a curative treatment for various hematopoietic diseases. HSPC function and behavior is tightly regulated by various cell types and factors in the bone marrow niche. One level of regulation comes from the sympathetic nervous system that innervates the niche and releases neurotransmitters to stromal cells. However, the direct regulation of HSPCs via cell surface expression of neural receptors has not been functionally explored. Using imaging mass spectrometry, we detected strong and regionally specific gamma-aminobutyric acid (GABA) neurotransmitter signal in the endosteal region of mouse bone marrow. GABBR1 is known to be expressed on human HSPCs (Steidl et al., Blood 2004), however its function in their regulation has not been investigated. Based on published mouse HSPC single cell RNA-seq data (Nestorowa et al., Blood 2016), we found that a subset of HSPCs expressed the GABA type B receptor subunit 1 (Gabbr1). We confirmed by surface receptor expression that a subset of mouse bone marrow HSPCs express Gabbr1 protein. Using the same single cell RNA-seq data as above, our own gene set enrichment analysis (GSEA) revealed positive correlation of Gabbr1 expression with genes involved in immune system processes, such as response to type I interferons. We generated a CRISPR-Cas9 Gabbr1 mutant mouse model on a C57/BL6 background suitable for hematopoietic studies. Analysis of Gabbr1 mutant bone marrow cells revealed a reduction in the absolute number of Lin-Sca1+cKit+ (LSK) HSPCs, but no change in the number of long-term hematopoietic stem cells (LT-HSCs). With further hematopoietic profiling, we discovered reduced numbers of white blood cells in peripheral blood that was primarily due to fewer B220+ cells. We show that Gabbr1 null HSPCs display reduced proliferative capacity, as well as diminished reconstitution ability when transplanted in a competitive setting. An in vitro differentiation assay revealed the impaired ability of Gabbr1 null HSPCs to produce B cell lineages. We tested our predicted association with type I interferon response by administration of poly(I:C) and found reduced HSPC proliferation in Gabbr1 null mice. Our results may translate well to humans, as a rare human SNP within the GABBR1 locus was found that correlates with altered leukocyte counts (Astle et al., Cell 2016). Our results indicate an important role for Gabbr1 in the regulation of HSPC proliferation and differentiation, highlighting Gabbr1 as an emerging factor in the modulation of HSPC function and behavior. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single Cell Transcriptomic Analysis of the Multiple Myeloma Bone Marrow Identifies a Unique Inflammatory Stromal Cell Population Associated with TNF Signaling

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 690-690
Author(s):  
Madelon M.E. de Jong ◽  
Zoltan Kellermayer ◽  
Natalie Papazian ◽  
M Duin ◽  
Annemiek Broyl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Single Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Research Funding ◽  
Stromal Cell ◽  
Gene Set Enrichment Analysis ◽  
Bone Marrow Niche ◽  
Cell Clusters

Background: In multiple myeloma, tumor cell survival, disease progression and therapy response are influenced by signals derived from the non-malignant bone marrow niche. This notwithstanding, a detailed in-vivo definition of the cells that define the multiple myeloma niche is lacking. Mesenchymal stromal cells are important niche constituents. Recent progress made with single cell transciptomics suggests that mesenchymal stromal cells are a dynamic population of cells that can exist as several subsets with functionally distinct activation and differentiation profiles. Aim: To identify mesenchymal stromal cell subsets specific for the multiple myeloma bone marrow niche, by comparing stromal cells from myeloma patients to non-cancer controls. Methods: The non-hematopoietic bone marrow niche was isolated from viably frozen bone marrow aspirates from 10 newly diagnosed multiple myeloma patients (6 hyperdiploid, 3 t(11;14) and 1 with deletion of 17p) and 2 non-cancer controls using high speed cell sorting. The purified cells were analyzed by 10X Genomics single cell sequencing directly post-thawing, without prior cell culture. From 10 multiple myeloma patients we generated single cell transcriptomes with an average read-depth of 20,000 reads per cell of in total 12,000 niche cells and from the 2 non-cancer controls a total of 3,500 niche cells. Transcriptomes were pooled and subjected to clustering analyses using the Seurat package for R to identify genetically distinct clusters of niche cells and changes in these clusters associated specifically with multiple myeloma. Results: The bioinformatical analyses generated 10 distinct clusters of niche cells, all of which were present in both non-cancer and multiple myeloma bone marrow. One of these clusters contained CDH5+ endothelial cells while the remaining 9 clusters were subsets of CXCL12+LEPR+ mesenchymal stromal cells. Because samples were taken from the central marrow by aspiration, peripheral endosteal or neuronal lineage cells were not represented in these clusters. Gene Set Enrichment Analysis (GSEA) of the stromal cell clusters from myeloma versus non-cancer controls revealed two significantly altered pathways: TNF signaling via NF-kB and Inflammatory response. Detailed analyses of the individual stromal cell clusters identified two clusters that were responsible for the inflammatory changes identified by GSEA. Both clusters were present in all myeloma patients, constituted on average 20% of total stromal cells and were defined by transcription of the inflammatory chemokines CXCL2, CXCL3 and CXCL8 the cytokine IL6. All these transcripts were absent from the equivalent clusters in control bone marrow. The presence of inflammatory stroma in the multiple myeloma niche indicates either the appearance of a novel stromal cell subset, or activation of pre-existing stromal cells. GSEA analyses suggested inflammatory signaling, and to functionally confirm this, we tested whether activation of stromal cells would induce the inflammatory stromal phenotype. Stimulation of primary human stromal cells in vitro with recombinant TNF was sufficient to induce transcription of CXCL2, CXCL3 and CXCL8, recapitulating the inflammatory transcriptome. Moreover, manual removal of these TNF target genes from the in-silico clustering analyses led to a merging of the inflammatory clusters with non-inflammatory clusters. This indicates that the major distinguishing feature of the myeloma-specific stromal cells are genes induced upon stromal cell activation. Conclusion: Through single cell transcriptomic analyses we have identified the presence of activated inflammatory stromal cells associated with TNF signaling in the multiple myeloma stromal niche. These inflammatory stromal cells are reminiscent of pathogenic cancer-associated fibroblasts found in solid tumors, where these cells create a pro-tumorigenic niche that favors tumor survival and proliferation while simultaneously inhibiting anti-cancer immunity. These findings represent the first description of myeloma-specific stromal cell subsets, and provide novel cellular targets for interventions aimed at disrupting the pro-tumorigenic microenvironment in multiple myeloma. Disclosures Broyl: Celgene, amgen, Janssen,Takeda: Honoraria. Sonneveld:Amgen: Honoraria, Research Funding; BMS: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; SkylineDx: Research Funding; Takeda: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding.

scholarly journals Disease Modeling and Phenotype Rescue Using Inducible Pluripotent Stem Cells from Patients with Diamond-Blackfan Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2496-2496
Author(s):  
Senthil Velan Bhoopalan ◽  
Min-Joon Han ◽  
Steven Ellis ◽  
Harry Lesmana ◽  
Jeremie H. Estepp ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
Wild Type ◽  
Dna Breaks ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Bone Marrow Failure Syndromes

Diamond-Blackfan anemia (DBA) is a congenital ribosomopathy and bone marrow failure syndrome manifesting typically in infancy with erythroid hypoplasia. Approximately half of affected individuals also have developmental anomalies. Over time, additional cytopenias can develop, including reduced hematopoietic stem and progenitor cells (HSPC). Heterozygous loss-of-function mutations in over 20 ribosomal protein (RP) genes cause approximately 70% of DBA cases, although only 7 genes (RPS19, RPL5, RPS26, RPL11, RPL35a, RPS24 and RPS7) account for over 90% of patients with a known DBA genotype. Medical therapies including steroids, chronic transfusions are partially effective but have considerable side effects. Hematopoietic stem cell transplantation (HSCT) from matched related or unrelated donors is curative with recently reported good outcomes, although many patients lack a suitable donor and/or have serious treatment-related comorbidities that increase HSCT-related toxicities. Case reports of spontaneous genetic reversion in DBA suggest that RP gene-corrected HSPC have competitive advantage over RP-deficient cells, thus providing the rationale for gene therapy as a feasible therapeutic approach. Induced pluripotent stem cell (iPSC) technology provides a robust model of human disease and can recapitulate hematopoietic defects encountered in bone marrow failure syndromes. The goals of this study was to establish a culture system from patient-derived iPSCs that can recapitulate key aspects of DBA pathophysiology and provide a preclinical model for gene manipulation to correct the abnormal phenotype (Figure 1). We developed iPSCs from individuals with DBA who were enrolled on INSIGHT (NCT02720679), an IRB-approved, prospective study that includes biobanking of peripheral blood mononuclear cells (PBMNC) from patients with bone marrow failure syndromes. We first reprogrammed these DBA PBMNC into iPSCs using non-integrating Sendai virus to establish lines with pathogenic mutations in RPS19 (c.191>T, p.Leu64Pro), RPS19 (c.184C>T, p.Arg62Trp), RPL11 (c.61dupT, p.Cys21Leufs*13), and a variant of uncertain significance (VUS) in RPS7 (c.277_279delGTC, pVal93del). Undifferentiated iPSC lines exhibited abnormal ribosomal biogenesis revealed by polysome profiling and pre-rRNA analysis. Upon in vitrodifferentiation to hematopoietic lineages, the mutant iPSCs recapitulated DBA phenotypes with reduced CD34+ HSPCs, near absence of erythroid colonies (BFU-E and CFU-E) colonies and failure to produce erythroid cells in liquid culture. We used two methods to correct single nucleotide RP mutations in DBA iPSCs (Figure 1): i) CRISPR/Cas9-mediated homology-directed repair, and ii) base-editing, which utilizes catalytically inactive Cas9 fused to a deaminase that interconverts nucleotides directly in the absence of double-stranded DNA breaks. Corrected "isogenic" lines showed phenotype similar to wild type controls, with restored erythroid differentiation, and normal polysome maturation and pre-rRNA ratios. Because some patients carry large intragenic or whole RP gene deletions that are not amenable to gene correction, we also explored the feasibility of gene rescue by inserting a wild type copy of the defective gene (Figure 1). Using zinc-finger nuclease (ZFN), we inserted wild type RP cDNA constructs into the "safe harbor" AAVS1 locus on chromosome 19, thereby rescuing abnormal phenotypes of patient-derived iPSC lines with RPS19(p.Arg62Trp) and RPL11(p.Cys21Leufs*34) mutations. Additionally, we explored lentiviral gene delivery as an alternative method for RP gene replacement. We compared different promoters including MND, PGK and EF1a and found that the latter was most effective at rescuing RP gene expression in iPSC cells. Transduction of lentiviral vectors with wild type RPS19or RPL11fused to the EF1a promoter into three iPSC lines with RPS19or RPL11mutations resulted in stable transgene expression of RPS19or RPL11genes and phenotypic rescue. This study supports the feasibility of establishing iPSCs from DBA subjects with different genotypes. These iPSC lines provide a useful resource for numerous studies of DBA including preclinical approaches to gene therapy, evaluating the pathogenicity of RP gene variants of unknown significance and examining the pathophysiology of RP haploinsufficiency. Disclosures Estepp: Esperion: Consultancy; Forma Therapeutics: Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Pfizer: Research Funding; Eli Lilly and Co: Research Funding; Daiichi Sankyo: Consultancy. Weiss:GlaxoSmithKline: Consultancy; Rubius Inc.: Consultancy; Cellarity Inc.: Consultancy; Beam Therapeutics: Consultancy; Esperion: Consultancy.

scholarly journals Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 368-368
Author(s):  
Jonathan Hoggatt ◽  
Pratibha Singh ◽  
Tiffany Tate ◽  
Peter V. Kharchenko ◽  
Amir Schajnovitz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Knockout Mice ◽  
Peripheral Blood ◽  
Research Funding ◽  
Gene Set Enrichment Analysis ◽  
Hematopoietic Stem ◽  
Equity Ownership

Abstract Hematopoietic stem cells (HSCs) are at the apex of lifelong blood cell production. Recent clonal analysis studies suggest that HSCs are heterogeneous in function and those that contribute to homeostatic production may be distinct from those that engraft during transplant. We developed a rapid mobilization regimen utilizing a unique CXCR2 agonist (an N-terminal truncated MIP-2a) and the CXCR4 antagonist AMD3100. A single subcutaneous injection of both agents together resulted in rapid mobilization in mice with a peak progenitor cell content in blood reached within 15 minutes. This mobilization was equivalent to a 5-day regimen of G-CSF. This rapid mobilization is the result of synergistic signaling, and was blocked in CXCR4 or CXCR2 knockout mice, confirming receptor and mechanism specificity. Mobilization is caused by synergistic release of MMP-9 from neutrophils and mobilization was blocked in MMP-9 knockout mice, mice treated with an anti-MMP-9 antibody, TIMP-1 transgenic mice, or mice where neutrophils were depleted in vivo using anti-GR-1 antibody. In vivo confocal imaging of mice demonstrated that the mobilization regimen causes a rapid and transient increase in bone marrow vascular permeability, "opening the doorway" for hematopoietic egress to the peripheral blood. Transplantation of 2x106 peripheral blood mononuclear cells (PBMC) from the rapid regimen resulted in a 4 or 6 day quicker recovery of neutrophils and platelets, respectively, compared to a G-CSF mobilized graft (n=12 mice per group, P<0.01). In limiting dilution competitive transplants, the rapid regimen demonstrated a greater than 2-fold enhancement in competitiveness (n=30 mice/treatment group, 2 individual experiments, P<0.001). Additionally, in secondarily transplanted mice, competitiveness of the rapidly mobilized graft increased as measured by contribution to chimerism, while G-CSF mobilized grafts remained static (n=16 mice/group, P<0.01). Surprisingly, despite robust enhancement in both short and long-term engraftment by the rapidly mobilized graft, phenotypic analysis of the blood of mobilized mice for CD150+ CD48- Sca-1+ c-kit+ Lineage neg (SLAM SKL) cells, a highly purified HSC population, showed lower numbers of phenotypically defined HSCs than in the G-CSF group. These data suggested that a unique subset of "highly engraftable" HSCs (heHSCs) are mobilized by the rapid regimen compared to G-CSF. However, as our earlier studies were performed using grafts that contained the total PBMC fraction (similar to the clinical apheresis product) we could not rule out the potential contribution of accessory cells to the enhanced engrafting ability of the heHSCs. Therefore, in 3 independent experiments, we mobilized large cohorts of mice with the rapid regimen or G-CSF and sorted SLAM SKL cells from the PBMC fraction and competitively transplanted equal numbers of SLAM SKL cells from either the rapid regimen or G-CSF and tracked contribution to chimerism over 36 weeks. Remarkably, the heHSCs from the rapid regimen demonstrated a 2-fold enhancement in competitiveness compared to SLAM SKL cells from the G-CSF group (n=17 mice/group, P<0.0004). While appreciation for HSC heterogeneity has grown, methods are lacking for prospectively isolating differing HSC populations with known biologic function, to study molecular heterogeneity. Like panning for gold, we sought to use the differential mobilization properties of our rapid regimen and G-CSF as a "biologic sieve" to isolate the heterogeneous HSC populations from the blood. We again flow sorted SLAM SKL cells from mice mobilized with the rapid regimen or G-CSF and performed RNA-Seq analysis of the purified populations. The heHSCs mobilized by the rapid regimen had a unique transcriptomic signature compared to G-CSF mobilized or random HSCs acquired from bone marrow (P<0.000001). Strikingly, gene set enrichment analysis (GSEA) demonstrated that the heHSCs had a gene signature highly significantly clustered to that of fetal liver HSCs, further demonstrating the selective harvesting of a subset of highly engraftable stem cells. Our results mechanistically define a new mobilization strategy, that in a single day can mobilize a graft with superior engraftment properties compared to G-CSF, and selectively mobilize a novel population of heHSCs with an immature molecular phenotype capable of robust long-term engraftment. Disclosures Hoggatt: Magenta Therapeutics: Consultancy, Equity Ownership, Research Funding. Scadden:Magenta Therapeutics: Consultancy, Equity Ownership; GlaxoSmithKline: Research Funding; Harvard University: Patents & Royalties. Pelus:GlaxoSmithKline: Consultancy.

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